Field
The invention relates generally to structural immunology, protein chemistry, and more particularly to in vitro protein folding.
Description of Related Art
The expression of recombinant proteins is important for the study of the biological functions of genes, the development of therapeutic drugs, and for industrial processes. There are several protein expression systems to produce the target proteins, such as bacteria, yeast, insect cells, mammalian cells, and cell-free systems. Expression in mammalian cells and insect cells produces biologically active proteins that contain post-translational modification(s), such as phosphorylation, acetylation, and glycosylation. However, expression using these systems gives low yields of the recombinant proteins, and the cost of these systems is generally expensive for industrial-scale or high throughput protein production. Cell-free systems also give low yields of recombinant proteins. Among bacterial systems, the Escherichia coli (E. coli) overexpression system is the most convenient and commonly used to produce recombinant proteins. However, expression of many proteins, particularly eukaryotic proteins, in E. coli leads to protein misfolding and aggregation to form inclusion bodies (Singh and Panda 2005, Burgess 2009, Seras-Franzoso, Peternel et al. 2015).
Recovering biologically active proteins at low cost is an important goal in protein folding from bacterial inclusion bodies, not only for analysis of the protein structure and function, but also for the development of therapeutic drugs and industrial processes for producing commercially relevant amounts of product. As inclusion bodies contain relatively pure and intact recombinant proteins, several approaches have been reported to recover these aggregated forms as biologically active proteins. In a typical procedure, aggregated forms are denatured and dissolved with a high concentration of denaturant, such as urea, guanidinium chloride (GdnHCl), or ionic detergents, such as N-lauroylsarcosine. These chemical reagents are used to decrease the non-covalent interactions between protein molecules. In addition, dithiothreitol or 2-mercaptoethanol is added to reduce undesirable inter- and/or intra-molecular disulfide bonds. Folding from denatured proteins (unfolded form) to the folded protein configurations necessary to provide biologically active proteins (folded form) occurs by the removal of denaturant. Folding efficiency (i.e., yield of folded protein) can be estimated by physical or biological activity, such as enzymatic activity, ligand binding, or spectroscopic methods if the folded state has a unique spectroscopic signal does not possess the folded protein configuration necessary to provide a biologically active protein
The procedure for the removal of the denaturant from denatured proteins is a key step in the efficient folding of proteins from denatured states. Several approaches have been reported, such as size-exclusion chromatography, reversed micelle systems, zeolite absorbing systems, and the natural GroEL-GroES chaperone system. These folding methods, using chromatographic or non-chromatographic strategies, have been described in recent reviews (Singh and Panda 2005, Burgess 2009, Yamaguchi and Miyazaki 2014, Seras-Franzoso, Peternel). Although these methods work well for many inclusion body proteins and denatured model proteins, in most cases there is a significant amount of protein precipitation, resulting in a low recovery yield. Therefore, the protein folding procedure is still performed with a series of trial-and-error folding experiments.
In dialysis, a chemically denatured protein is folded by sufficiently decreasing the denaturant concentration, permitting folding. One-step dialysis (high denaturant concentration with respect to the folding buffer) is a simple method. The protein concentration remains nearly constant during the procedure. As the concentration of denaturant decreases with increased dialysis time (until equilibrium is reached), the amount of folded protein likewise increases. However, misfolding and protein aggregation will also occur, possibly due to contact between exposed hydrophobic surfaces. This suggests that a rapid decrease in denaturant concentration can initiate the formation of aggregates.
To solve this problem, step-wise dialysis has been used. In step-wise dialysis, the denatured proteins are first dialyzed to equilibrium against a high denaturant concentration, then dialyzed against a lower denaturant concentration until equilibrium is reached, and, then dialyzed against an even lower denaturant concentration. This step-wise dialysis against lower denaturant concentrations may be repeated multiple times. Such gradual removal of denaturant from the denatured proteins can achieve high folding efficiency (Yamaguchi and Miyazaki 2014). However, it is a time-consuming procedure, typically requiring multiple days. In addition, at medium denaturant concentrations the proteins can misfold or aggregate. Protein aggregation therefore remains a significant technical problem associated with the production of proteins.
The dilution method is a simple procedure used for protein folding. The denatured proteins are directly diluted multiple times with a folding buffer that does contains progressively lower or no denaturants. In the dilution method, the protein concentration is also decreased. As aggregation is a function of the protein concentration, a low protein concentration should help avoid intermolecular aggregation during the procedure. However, because proteins diffuse slower than denaturant, diluted proteins are likely to very quickly aggregate, similar to what is observed in one-step dialysis. In addition, this method requires a large volume of buffer, increasing cost and decreasing utility. Moreover, difficulties can be encountered in uniformly mixing large volumes, wherein reformation of aggregates can occur.
The addition of chemical additives, such as denaturants, protein stabilizers, and protein aggregation inhibitors, has been described to prevent protein aggregation. Urea and GdnHCl are typical protein denaturants. At high concentrations, denaturants will denature proteins by the chaotropic effect and/or by interacting with the unfolded state, while at low denaturant concentrations, some denaturants have been reported to stabilize the structure of the target protein by inhibiting/destabilizing aggregates (Yamaguchi and Miyazaki 2014). (NH4)2SO4 is a protein stabilizer that can stabilize protein structure at low concentration through electrostatic interactions, which changes the solubility of the native (folded) structure protein. However, protein destabilizers often accelerate protein aggregation. Arginine and its derivatives are among some of the amino acids classified as protein aggregation inhibitors. These amino acids are often used in the folding process, and are reported to increase yields by decreasing aggregation (Yamaguchi and Miyazaki 2014).
Some investigators have proposed microfluidics approaches as rapid and simple protein folding methods. In a microfluidic system, the laminar flow in microchannels is used to create a well-defined and predictable interfacial region. However, difficult-to-fold proteins can aggregate in the microchannels due to rapid removal of denaturant from the denatured proteins. Thus, microfluidic chips with rapid mixing are not always useful in the folding of difficult-to-fold proteins (Yamaguchi and Miyazaki 2014).
A need continues to exist in the art of pharmaceutical drug production and other technical areas related to protein/peptide recovery for more efficient methods for providing properly folded proteins. In particular, improved methods to obtain properly folded proteins from proteins within inclusion bodies (or other sources of protein that exist in an aggregated form), and methods that may be implemented in protein recycling techniques, remain needed. In addition, a reproducible and reliable method for producing a sufficient amount of important, yet difficult to fold complex protein assemblages, such as major histocompatability complex (MHC) proteins, their complexes with peptides and multimeric peptide/MHC complexes, is needed for facilitating drug/pharmaceutical development, immune disease treatment modalities, and biotechnological reagents.